Cytosporinols A-C, new caryophyllene sesquiterpenoids from Cytospora sp.

Three new caryophyllene sesquiterpenoids, cytosporinols A-C (1–3), have been isolated from solid cultures of Cytospora sp. The structures of 1–3 were elucidated primarily by NMR spectroscopy, and 3 was further confirmed by X-ray crystallography. The absolute configurations of the C-11 secondary alcohol in 1 and the 6,8-diol moiety in 3 were deduced using the modified Mosher and Snatzke’s method, respectively. Compounds 2 and 3 showed moderate cytotoxicity against HeLa cells.


Introduction
Caryophyllene sesquiterpenoids have been isolated frequently from fungi as the bioactive principles. Examples include the pestalotiopsins, immunosuppressive and cytotoxic agents produced by the endophyte Pestalotiopsis sp. isolated from Taxus brevifolia; 1,2 pestalotiopsolide A, teadolidol, and 6-epiteadolidol, highly oxidized caryophyllenes from a Pestalotiopsis sp. endophytic to the bark of Pinus taeda; 3 the 6-hydroxpunctaporonins, antibacterial metabolites from a fungicolous Pestalotiopsis disseminata; 4 fuscoatrol A, a cytotoxic agent from a marine-derived Humicola fuscoatra KMM 4629; 5 the punctaporonins (also named as punctatins), antifungal agents from a coprophilous Poronia punctata; [6][7][8][9][10][11] walleminol and walleminone, two cis-fused iso-caryophyllenes from the toxigenic fungus Wallemia sebi; 12 and Sch 725432, 601253, 601254, and 725434, antifungal agents from Chrysosporium pilosum. 13 During an ongoing search for new bioactive natural products from fungi of unique habitats, we initiated chemical studies of the fungi either inhabiting the fruiting body and larvae of Cordyceps sinensis 14 or its surface soil. 15 As an extension, we also studied those species isolated from the soil samples that were collected on the Qinghai-Tibetan plateau at altitudes above 3,200 m, the environment in which Cordyceps sinensis was typically found. Our initial investigation of such an ascomycetous fungus Cytospora sp. led to the isolation of three antimicrobial caryophyllene-derived meroterpenoids. 16 Since the HPLC chromatogram of the crude extract revealed the presence of some minor components that could be the cytotoxic principles remained to be identified, the fungus was refermented in a larger scale using the same solid fermentation culture medium in which the meroterpenoids were first isolated. 16 Fractionation of an EtOAc extract afforded three new caryophyllene sesquiterpenoids which we named cytosporinols A-C (1-3). Details of the isolation, structure elucidation, and cytotoxicity of these compounds are described herein.

Results and Discussion
Cytosporinol A (1) was assigned the molecular formula C 16 H 24 O 4 (five degrees of unsaturation) by HRESIMS. Analysis of its NMR data (Table 1) revealed two exchangeable protons (δ H 3.37 and 3.93, respectively), four methyl groups (one O-methyl), two methylenes, six methines including four oxymethines, two sp 3 quaternary carbons (one oxygenated), and one tetrasubstituted olefin. These data accounted for all the 1 H and 13 C NMR resonances, suggesting that 1 was a tetracyclic compound. The 1 H-1 H COSY NMR data showed *To whom correspondence should be addressed. E-mail: cheys@im.ac.cn (Y.S. Che); cuicb@126.com (C.B. Cui). two isolated spin-systems of C-10-C-1-C-3 (including OH-11 and C-12) and C-6-C-7 (including OH-6). Interpretation of the HMBC data of 1 established a cyclobutane partial structure with two methyl groups attached to C-4. HMBC cross-peaks from H-6 to C-2 and from H 2 -7 to C-5 indicated that C-6 is attached to the cyclobutane ring at C-5. The H 2 -7 protons were correlated to C-8, C-9, and C-15, while the Me-15 protons were correlated to C-7, C-8, and C-9 in the HMBC spectrum of 1, linking the C-8 olefinic carbon (δ C 130.2) to C-7, C-9 (δ C 134.5), and C-15. HMBC correlations from H-10 to C-8 and C-12, H-11 to C-9, and from H-12 to C-8 connected C-9 to both C-10 and C-12, completing the partial structure with a cyclopentane moiety fused to a cyclooctene unit at C-1/C-9.
The key HMBC correlation from H-12 to C-5 (δ C 95.5) connected both C-5 and C-12 (δ C 89.6) to the same oxygen atom to form a tetrahydrofuran (THF) ring fused to the cyclobutane and cyclopentane moieties at C-2/C-5 and C-1/C12, respectively. The HMBC correlation from the Omethyl proton signal Me-16 (δ H 3.12) to the C-10 oxymethine carbon (δ C 86.4) located the only methoxy group at C-10. On the basis of these data, the planar structure of 1 was established.
The relative configuration of 1 was deduced by analysis of the 1 H-1 H coupling constants and NOESY data ( Figure 1). The vicinal coupling constants observed for H-1 with H-11 and H-12 were 7.4 and 6.5 Hz, respectively, suggesting their cis-relative orientation, 17 consistent with a similar THF moiety found in the known compound punctaporonin C (4). 9,18 NOESY correlations of Me-13 with H-2, H-3a, and H-6 indicated that these protons are all on the same face of the ring system, whereas those of H-3b with H-1 and Me-14, and of H-12 with Me-16 were used to place them on the opposite face. Collectively, these data allowed assignment of the relative configuration of 1. The 1S, 2R, 5S, 6S, 10R, 11R, and 12S absolute configurations were deduced for the stereogenic centers in 1 using the modified Mosher method 19 as illustrated in Figure 2, which were also consistent with those assigned for corresponding stereogenic centers in 4. 18 Cytosporinol B (2) was assigned the same molecular formula C 16 H 24 O 4 as 1 by HRESIMS. Analysis of its NMR data ( Table 2) revealed structural fragments similar to those presented in 1. However, the C-8-C-15 unit and the C-9 olefinic carbon (δ C 134.5) were replaced by a terminal olefin (δ H /δ C 4.87/117.3, 144.7) and a methine (δ H /δ C 2.90/58.0), respectively, which were supported by relevant 1 H-1 H COSY and HMBC correlations. Therefore, the planar structure of 2 was proposed as shown. The relative configuration of 2 was also determined by analysis of the 1 H-1 H coupling constants and NOESY data ( Figure 1). The coupling patterns and NOESY correlations for the relevant protons in 2 were nearly identical to those of 1, indicating that they possess the same relative configuration, except for the additional stereogenic center C-9, which was assigned by comparison of the coupling constant between H-9 and H-12 (9.7 Hz) with that reported for 4. 9,18 This assignment was supported by NOESY correlations of H-9 with H-1 and Me-16. Considering the absolute configuration established for 1 by the modified Mosher method, C-9 was deduced to have the S configuration.

Experimental Section
General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a Shimadzu Biospec-1601 spectrophotometer. CD spectra were recorded on a JASCO J-815 spectropolarimeter. IR data were recorded using a Nicolet Magna-IR 750 spectrophotometer. 1 H and 13 C NMR data were acquired with Varian Mercury-400 and -500 spectrometers using solvent signals (acetone-d 6 April, 2007. The isolation and identification of the strain have been previously described. 16a The fungus was cultured on slants of PDA at 25 °C for 10 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions, 15 pieces were used to inoculate in three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5 and sterilized by autoclave. Three flasks of the inoculated media were incubated at 25 °C on a rotary shaker at 170 rpm for five days to prepare the seed culture. Spore inoculum was prepared by suspending the seed culture in sterile, distilled H 2 O to give a final spore/cell suspension of 1 × 10 6 /mL determined by microscope and hemocytometer. Fermentation was carried out in 12 Fernbach flasks (500 mL) each containing 80 g of rice. Distilled H 2 O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 °C for 40 days.

Preparation of (R)-(1a) and (S)-MTPA (1b) Esters.
Absolute Configuration of the 6,8-Diol Moiety in 3. 20 HPLC grade DMSO was dried with 4 Å molecular sieves. According to a published procedure, a mixture of 1:2 diol/Mo 2 (OAc) 4 for 3 was subjected to CD measurements at a concentration of 0.6 mg/mL. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until stationary (about 10 min after mixing). The inherent CD was subtracted. The observed sign of the diagnostic band at around 400 nm in the induced CD spectrum was correlated to the absolute configuration of the 6,8-diol moiety.
MTS Assay. 28 The assay was run in triplicate. In a 96-well plate, each well was plated with 2-5 × 10 3 cells (depending on the cell multiplication rate). After cell attachment overnight, the medium was removed, and each well was treated with 50 µL medium containing 0.1% DMSO, or appropriate concentrations of the test compounds and the positive control cisplatin (100 mM as stock solution of a compound in DMSO and serial dilutions; the test compounds showed good solubility in DMSO and did not precipitate when added to the cells). The plate was incubated for 72 h at 37 °C in a humidified, 5% CO 2 atmosphere. Proliferation assessed by adding 20 μL of MTS (Promega) to each well in the dark, followed by a 90 min incubation at 37 °C. The assay plate was read at 490 nm using a microplate reader.

Electronic Supplementary Material
Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-012-0018-z and is accessible for authorized users.